Capacitation

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Capacitation is the penultimate [1] step in the maturation of mammalian spermatozoa and is required to render them competent to fertilize an oocyte. [2] This step is a biochemical event; the sperm move normally and look mature prior to capacitation. In vivo , capacitation occurs after ejaculation, when the spermatozoa leave the vagina and enter the upper female reproductive tract. The uterus aids in the steps of capacitation by secreting sterol-binding albumin, lipoproteins, and proteolytic and glycosidasic enzymes such as heparin.

Contents

For purposes of in vitro fertilization, capacitation occurs by incubating spermatozoa that have either undergone ejaculation or have been extracted from the epididymis and incubated in a defined medium for several hours. There are different techniques to perform the capacitation step: simple washing, migration (swim-up), density gradients, and filter. The objective is to isolate as many motile spermatozoa as possible and to eliminate non-motile or dead spermatozoa. After either in vivo or in vitro capacitation the sperm must undergo the final maturation step, activation, involving the acrosome reaction.

Non-mammalian spermatozoa do not require this capacitation step and are ready to fertilize an oocyte immediately after release from the male.

Function and mechanism

Capacitation has two effects: destabilisation of the acrosomal sperm head membrane which allows it to penetrate the outer layer of the egg, and chemical changes in the tail that allow a greater mobility in the sperm. [3] The changes are facilitated by the removal of sterols (e.g. cholesterol) and non-covalently bound epididymal/seminal glycoproteins. The result is a more fluid membrane with an increased permeability to Ca2+ ion.

An influx of Ca2+ produces increased intracellular cAMP levels and thus, an increase in motility. Hyperactivation coincides with the onset of capacitation and is the result of the increased Ca2+ levels. It has a synergistic stimulatory effect with adenosine that increases adenylyl cyclase activity in the sperm.[ citation needed ]

The tripeptide fertilization promoting peptide (FPP) is essential for controlling capacitation. FPP is produced in the prostate gland as a component of the seminal fluid. FPP comes into contact with the spermatozoa during ejaculation, as the sperm and seminal fluid mix. High levels of active FPP prevent capacitation. After ejaculation, the concentration of FPP drops in the female reproductive tract. [ citation needed ]

Induction

Because assisted reproductive technologies, or ARTs, such as in vitro fertilization (IVF) or intrauterine insemination (IUI) require the induction of sperm cell capacitation outside of normal biological parameters, numerous methods have been developed to induce this process in mammalian sperm cells. Sperm cells are harvested through ejaculation or harvested from the caudal epididymis and allowed to liquefy at room temperature. Capacitation can then be induced by adding media designed to mimic the electrolytic composition of the fallopian tubes, where fertilization occurs. These media vary between species, but are saline-based and contain energy substrates such as lactate, pyruvate, and possibly glucose. A cholesterol acceptor is required to facilitate the removal of cholesterol from the sperm cell membrane, which is always albumin. Bovine serum albumin is typically used for in vitro animal studies, and human serum albumin (HSA) is used in human sperm capacitation induction.

Bicarbonate is a vital component of capacitation-inducing media, as it is co-transported into the cytosol where it activates soluble adenylyl cyclase (sAC) as well as acts as a pH buffer necessary to prevent decreasing the pH in the culture, a necessary addition when incubating cells at 5% CO2 as is generally used although not required. Calcium chloride is added to facilitated the influx via of calcium cations. [4] [5] In animal models, Tyrode's albumin lactate pyruvate (TALP) medium is typically used as a base, which contains each of these components. In humans, human tubal fluid (HTF) is used.

These media can be supplemented with other chemicals to induce hyperactivated sperm motility and/or the acrosome reaction. For animal in vitro fertilization, caffeine at 5 mM concentration is a strong inducer of sperm capacitation in vitro. [6] [7] Calcium ionophores are also ideal to induce capacitation. [7] Adding heparin to capacitation inducing medium mimics the secretion of heparin-like gycosaminoglycans (GAGs) near the oocyte and initiates the acrosome reaction. This effect is magnified when adding lysophosphatidylcholine (LC) in conjunction with heparin. [8] Catecholamines such as norepinephrine at low concentrations have been shown to assist in acrosome reaction induction. [9]

In vitro capacitation techniques

The traditional methods to perform in vitro capacitation are:

However, nowadays there are new techniques which surpass these ones. For instance, we have PICSI, MACS or microfluidic chips.

Measurement

Numerous methods have been developed to assess the degree to which sperm cells are undergoing capacitation in vitro. Computer-aided sperm analysis (CASA) was developed in the 1980s for measuring sperm kinematics. [11] CASA uses phase-contrast microscopy combined with sperm tracking software to analyze sperm motility parameters. [11] Certain parameters such as curvilinear velocity (VCL), straightline velocity (VSL), average path velocity (VAP), and the amplitude of lateral head displacement (ALH) have been shown to be positively correlated with the acquisition of fertilization competency and are thus used to identify hyperactive sperm cell motility. [12]

While motility measurements are critical for identifying the presence of hyperactive motility, additional methods have been developed to identify the occurrence of the acrosome reaction. A simple method uses Coomassie brilliant blue G250 to stain cells, providing visual evidence of intact or reacted acrosomes. [13] More advanced techniques employ fluorescent or electron microscopy methods. Fluorescein-conjugated Peanut agglutinin (FITC-PNA) or Pisum sativum agglutinin (FITC-PSA) can be used to fluorescently tag the acrosome of sperm cells, which can be then used to assess the status of the acrosome using a fluorescent microscope. [14] [15] [16]

Discovery

The discovery of this process was independently reported in 1951 by both Min Chueh Chang [17] and Colin Russell Austin. [18] [19]

See also

Related Research Articles

<span class="mw-page-title-main">Spermatozoon</span> Motile sperm cell

A spermatozoon is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote.

<span class="mw-page-title-main">Fertilisation</span> Union of gametes of opposite sexes during the process of sexual reproduction to form a zygote

Fertilisation or fertilization, also known as generative fertilisation, syngamy and impregnation, is the fusion of gametes to give rise to a zygote and initiate its development into a new individual organism or offspring. While processes such as insemination or pollination, which happen before the fusion of gametes, are also sometimes informally referred to as fertilisation, these are technically separate processes. The cycle of fertilisation and development of new individuals is called sexual reproduction. During double fertilisation in angiosperms, the haploid male gamete combines with two haploid polar nuclei to form a triploid primary endosperm nucleus by the process of vegetative fertilisation.

<span class="mw-page-title-main">Intracytoplasmic sperm injection</span> In vitro fertilization procedure

Intracytoplasmic sperm injection is an in vitro fertilization (IVF) procedure in which a single sperm cell is injected directly into the cytoplasm of an egg. This technique is used in order to prepare the gametes for the obtention of embryos that may be transferred to a maternal uterus. With this method, the acrosome reaction is skipped.

<span class="mw-page-title-main">Acrosome</span>

The acrosome is an organelle that develops over the anterior (front) half of the head in the spermatozoa of humans, and many other animals. It is a cap-like structure derived from the Golgi apparatus. In placental mammals, the acrosome contains degradative enzymes. These enzymes break down the outer membrane of the ovum, called the zona pellucida, allowing the haploid nucleus in the sperm cell to join with the haploid nucleus in the ovum. This shedding of the acrosome, or acrosome reaction, can be stimulated in vitro by substances a sperm cell may encounter naturally such as progesterone or follicular fluid, as well as the more commonly used calcium ionophore A23187. This can be done to serve as a positive control when assessing the acrosome reaction of a sperm sample by flow cytometry or fluorescence microscopy. This is usually done after staining with a fluoresceinated lectin such as FITC-PNA, FITC-PSA, FITC-ConA, or fluoresceinated antibody such as FITC-CD46.

<span class="mw-page-title-main">Epididymis</span> Tube that connects a testicle to a vas deferens

The epididymis is an elongated tubular structure attached to the posterior side of each one of the two male reproductive glands, the testicles. It is a single, narrow, tightly coiled tube in adult humans, 6 to 7 centimetres in length; uncoiled the tube would be approximately 6 m long. It connects the testicle to the vas deferens in the male reproductive system. The epididymis serves as an interconnection between the multiple efferent ducts at the rear of a testicle (proximally), and the vas deferens (distally). Its primary function is the storage, maturation and transport of sperm cells.

<span class="mw-page-title-main">Acrosome reaction</span> Sperm-meets-egg process

During fertilization, a sperm must first fuse with the plasma membrane and then penetrate the female egg cell to fertilize it. Fusing to the egg cell usually causes little problem, whereas penetrating through the egg's hard shell or extracellular matrix can be more difficult. Therefore, sperm cells go through a process known as the acrosome reaction, which is the reaction that occurs in the acrosome of the sperm as it approaches the egg.

Hyperactivation is a type of sperm motility. Hyperactivated sperm motility is characterised by a high amplitude, asymmetrical beating pattern of the sperm tail (flagellum). This type of motility may aid in sperm penetration of the zona pellucida, which encloses the ovum.

<span class="mw-page-title-main">Acrosin</span> Mammalian protein found in Homo sapiens

Acrosin is a digestive enzyme that acts as a protease. In humans, acrosin is encoded by the ACR gene. Acrosin is released from the acrosome of spermatozoa as a consequence of the acrosome reaction. It aids in the penetration of the Zona Pellucida.

<span class="mw-page-title-main">Sperm</span> Male reproductive cell in anisogamous forms of sexual reproduction

Sperm is the male reproductive cell, or gamete, in anisogamous forms of sexual reproduction. Animals produce motile sperm with a tail known as a flagellum, which are known as spermatozoa, while some red algae and fungi produce non-motile sperm cells, known as spermatia. Flowering plants contain non-motile sperm inside pollen, while some more basal plants like ferns and some gymnosperms have motile sperm.

<span class="mw-page-title-main">Spermiogenesis</span> Final stage of spermatogenesis, involving spermatid maturation

Spermiogenesis is the final stage of spermatogenesis, during which the spermatids develop into mature spermatozoa. At the beginning of the stage, the spermatid is a more or less circular cell containing a nucleus, Golgi apparatus, centriole and mitochondria; by the end of the process, it has radically transformed into an elongated spermatozoon, complete with a head, midpiece, and tail.

<span class="mw-page-title-main">Human fertilization</span> Union of a human egg and sperm

Human fertilization is the union of an egg and sperm, occurring primarily in the ampulla of the fallopian tube. The result of this union leads to the production of a fertilized egg called a zygote, initiating embryonic development. Scientists discovered the dynamics of human fertilization in the 19th century.

Male infertility refers to a sexually mature male's inability to impregnate a fertile female. In humans, it accounts for 40–50% of infertility. It affects approximately 7% of all men. Male infertility is commonly due to deficiencies in the semen, and semen quality is used as a surrogate measure of male fecundity. More recently, advance sperm analyses that examine intracellular sperm components are being developed.

Decapacitation factor (DF) is composed of sperm surface-associated proteins which modulate the fertilizing ability of spermatozoa. Decapacitation is a reversible process that converts fertile, capacitated sperm to less-fertile uncapacitated sperm. This activity is achieved by interaction between cholesterol, phospholipids and fibronectin-like substances and delivered via small vesicles in seminal plasma. DF prevents onset of capacitation. Many DFs are released in secretions from the epididymis and accessory organs of the male reproductive system. However, some DFs have been identified that are located on the acrosome of sperm. Normally, capacitation is initiated through the loss of DF before the spermatozoa can perform the acrosomal reaction. Physiologically decapacitation will inhibit the acrosomal reaction as DFs reassociate onto the sperm surface. For example, one way this can be achieved is through spermatozoal membrane stabilization by maintaining physiological cholesterol/phospholipid ratio.

The hamster zona-free ovum test, or hamster egg-penetration test, or sometimes just hamster test, is an in-vitro test used to study physiological profile of spermatozoa. The primary application of the test is to diagnose male infertility caused by sperm unable to penetrate the ova. The test has limited value, due to expense and a high false negative rate.

<span class="mw-page-title-main">Semen analysis</span> Scientific analysis of semen

A semen analysis, also called seminogram or spermiogram, evaluates certain characteristics of a male's semen and the sperm contained therein. It is done to help evaluate male fertility, whether for those seeking pregnancy or verifying the success of vasectomy. Depending on the measurement method, just a few characteristics may be evaluated or many characteristics may be evaluated. Collection techniques and precise measurement method may influence results.

<span class="mw-page-title-main">Sperm motility</span> Process involved in the controlled movement of a sperm cell

Sperm motility describes the ability of sperm to move properly through the female reproductive tract or through water to reach the egg. Sperm motility can also be thought of as the quality, which is a factor in successful conception; sperm that do not "swim" properly will not reach the egg in order to fertilize it. Sperm motility in mammals also facilitates the passage of the sperm through the cumulus oophorus and the zona pellucida, which surround the mammalian oocyte.

Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. Several conventional techniques of centrifugation or swim-up. Newly applied methods such as flow cytometry expand the possibilities of sperm sorting and new techniques of sperm sorting are being developed.

Sperm guidance is the process by which sperm cells (spermatozoa) are directed to the oocyte (egg) for the aim of fertilization. In the case of marine invertebrates the guidance is done by chemotaxis. In the case of mammals, it appears to be done by chemotaxis, thermotaxis and rheotaxis.

Semen cryopreservation is a procedure to preserve sperm cells. Semen can be used successfully indefinitely after cryopreservation. It can be used for sperm donation where the recipient wants the treatment in a different time or place, or as a means of preserving fertility for men undergoing vasectomy or treatments that may compromise their fertility, such as chemotherapy, radiation therapy or surgery. It is also often used by trans women prior to medically transitioning in ways that affect fertility, such as feminizing hormone therapy and orchiectomies.

Antisperm antibodies (ASA) are antibodies produced against sperm antigens.

References

  1. ↑ Johnson MH (2007). Essential reproduction (6th ed.). Malden Massachusetts: Blackwell Scientific Publications. pp. 177–178. ISBN   978-1-4051-1866-8.
  2. ↑ Lozano GM, Bejarano I, Espino J, González D, Ortiz A, García JF, Rodríguez AB, Pariente JA (2009). "Density gradient capacitation is the most suitable method to improve fertilization and to reduce DNA fragmentation positive spermatozoa of infertile men". Anatolian Journal of Obstetrics & Gynecology. 3 (1): 1–7.
  3. ↑ Okabe M (2013). "The cell biology of mammalian fertilization". Development. 140 (22): 4471–4479. doi: 10.1242/dev.090613 . PMID   24194470. S2CID   2015865.
  4. ↑ Visconti PE, Galantino-Homer H, Moore GD, Bailey JL, Ning X, Fornes M, Kopf GS (1998). "The molecular basis of sperm capacitation". Journal of Andrology. 19 (2): 242–248. doi: 10.1002/j.1939-4640.1998.tb01994.x . PMID   9570749. S2CID   14590131.
  5. ↑ Puga Molina LC, Luque GM, Balestrini PA, Marín-Briggiler CI, Romarowski A, Buffone MG (2018). "Molecular Basis of Human Sperm Capacitation". Frontiers in Cell and Developmental Biology. 6: 72. doi: 10.3389/fcell.2018.00072 . PMC   6078053 . PMID   30105226.
  6. ↑ Nabavi N, Todehdehghan F, Shiravi A (September 2013). "Effect of caffeine on motility and vitality of sperm and in vitro fertilization of outbreed mouse in T6 and M16 media". Iranian Journal of Reproductive Medicine. 11 (9): 741–746. PMC   3941327 . PMID   24639814.
  7. 1 2 Barakat IA, Danfour MA, Galewan FA, Dkhil MA (2015). "Effect of various concentrations of caffeine, pentoxifylline, and kallikrein on hyperactivation of frozen bovine semen". BioMed Research International. 2015: 948575. doi: 10.1155/2015/948575 . PMC   4407405 . PMID   25950005.
  8. ↑ Parrish JJ, Susko-Parrish J, Winer MA, First NL (1988). "Capacitation of bovine sperm by heparin". Biology of Reproduction. 38 (5): 1171–1180. doi: 10.1095/biolreprod38.5.1171 . PMID   3408784.
  9. ↑ Way AL, Killian GJ (2002). "Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine". Journal of Andrology. 23 (3): 352–357. doi:10.1002/j.1939-4640.2002.tb02242.x. PMID   12002437.
  10. ↑ "Sperm Swim up – Percoll | Fertilitycrete".
  11. 1 2 Mortimer D, Mortimer ST (2013). "Computer-Aided Sperm Analysis (CASA) of sperm motility and hyperactivation". Spermatogenesis. Methods in Molecular Biology. Vol. 927. pp. 77–87. doi:10.1007/978-1-62703-038-0_8. ISBN   978-1-62703-037-3. PMID   22992905.
  12. ↑ Verstegen J, Iguer-Ouada M, Onclin K (2002). "Computer assisted semen analyzers in andrology research and veterinary practice". Theriogenology. 57 (1): 149–179. doi:10.1016/S0093-691X(01)00664-1. PMID   11775967.
  13. ↑ Lu HY, Lu JC, Hu YA, Wang YM, Huang YF (2002). "[Detection of human sperm morphology and acrosome reaction with Coomassie brilliant blue staining]". Zhonghua Nan Ke Xue = National Journal of Andrology. 8 (3): 204–206. PMID   12478845.
  14. ↑ Cheng FP, Fazeli A, Voorhout WF, Marks A, Bevers MM, Colenbrander B (1996). "Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa". Journal of Andrology. 17 (6): 674–682. doi:10.1002/j.1939-4640.1996.tb01852.x. PMID   9016398.
  15. ↑ Lybaert P, Danguy A, Leleux F, Meuris S, Lebrun P (2009). "Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa". Histology and Histopathology. 24 (8): 999–1007. doi:10.14670/HH-24.999. PMID   19554507.
  16. ↑ Ozaki T, Takahashi K, Kanasaki H, Miyazaki K (2002). "Evaluation of acrosome reaction and viability of human sperm with two fluorescent dyes". Archives of Gynecology and Obstetrics. 266 (2): 114–117. doi:10.1007/s004040000112. PMID   12049293. S2CID   19898325.
  17. ↑ Chang MC (1951). "Fertilizing capacity of spermatozoa deposited into the fallopian tubes". Nature. 168 (4277): 697–698. Bibcode:1951Natur.168..697C. doi:10.1038/168697b0. PMID   14882325. S2CID   4180774.
  18. ↑ Austin CR (1951). "Observations on the penetration of the sperm in the mammalian egg". Australian Journal of Scientific Research B. 4 (4): 581–596. doi: 10.1071/BI9510581 . PMID   14895481.
  19. ↑ "Obituary: Colin Austin" (PDF). Australian Academy of Science Newsletter. 60: 11. 2004. Archived from the original (PDF) on 2008-07-19. Retrieved 2008-07-16.

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